title: Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.
abstract: Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins.
authors.affiliation: Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Branigan
authors.affiliation: Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK.
authors.forename: Christos
authors.initials: C
authors.lastname: Pliotas
authors.affiliation: Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK.
authors.forename: Gregor
authors.initials: G
authors.lastname: Hagelueken
authors.affiliation: Biomedical Sciences Research Complex, University of St. Andrews, St. Andrews, UK.
authors.forename: James H
authors.initials: JH
authors.lastname: Naismith
mesh_terms: D003545:Cysteine / Q000032:analysis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000032:analysis*; D008565:Membrane Proteins / Q000737:chemistry*; D055550:Protein Stability; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
chemical_list: D005456:Fluorescent Dyes; D008565:Membrane Proteins; D011506:Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine